Abstract:

The present invention is related to the discovery that the detection of
one or more biomarkers in a body sample can identify hematapoietic
progenitors that are precursors to a specific blood cell lineage. The
methods of the present invention are also directed to the detection of
the dysregulation of these biomarkers as a diagnostic assay for the
certain disease states, such as cancer.

Claims:

1. A method of directing the differentiation of a hematopoietic stem cell
(HSC), a hematopoietic progenitor cell (HPC), or a combination thereof,
said method comprising introducing into said cell a zinc finger protein
(Zfp105), wherein said Zfp105 upregulates expression of a natural killer
(NK) cell-specific polynucleotide in said cell, thereby inducing
differentiation of said cell into an NK cell.

2. The method of claim 1, wherein said Zfp105 is delivered to said cell as
a polypeptide.

3. The method of claim 1, wherein said Zfp105 is delivered to said cell as
a nucleic acid.

4. The method of claim 3, wherein said nucleic acid is contained within a
vector.

5. The method of claim 4, wherein said vector is a viral vector.

6. The method of claim 5, wherein said viral vector is selected from the
group consisting of a retroviral vector, an adenoviral vector, and an
adeno-associated viral vector.

7. The method of claim 4, wherein said vector is a non-viral vector.

8. The method of claim 1, wherein said cell is human.

9. A method of directing the differentiation of a HSC, a HPC, or a
combination thereof, said method comprising introducing into said cell an
Ets2, wherein said Ets2 upregulates expression of a monocyte-specific
polynucleotide in said cell thereby inducing differentiation of said cell
into a monocyte.

10. The method of claim 9, wherein said Ets2 is delivered to said cell as
a polypeptide.

11. The method of claim 9, wherein said Ets2 is delivered to said cell as
a nucleic acid.

12. The method of claim 11, wherein said nucleic acid is contained within
a vector.

13. The method of claim 12, wherein said vector is a viral vector.

14. The method of claim 13, wherein said viral vector is selected from the
group consisting of a retroviral vector, an adenoviral vector, and an
adeno-associated viral vector.

15. The method of claim 12, wherein said vector is a non-viral vector.

16. The method of claim 9, wherein said cell is human.

17. A method of directing the differentiation of HSC, a HPC, or a
combination thereof, to a B-cell, said method comprising introducing to
said cell at least one biomarker selected from the list consisting of
Chd7 (chromodomain helicase DNA binding protein 7), Edaradd (EDAR
(ectodysplasin-A receptor)-associated death domain, 2210016F16Rik, Dzip1
(DAZ interacting protein 1), and Tbl1x (transducin (beta)-like 1
X-linked).

18. The method of claim 17, wherein said biomarker is delivered to said
cell as a polypeptide.

19. The method of claim 17, wherein said biomarker is delivered to said
cell as a nucleic acid.

20. The method of claim 19, wherein said nucleic acid is contained within
a vector.

21. The method of claim 20, wherein said vector is a viral vector.

22. The method of claim 21, wherein said viral vector is selected from the
group consisting of a retroviral vector, an adenoviral vector, and an
adeno-associated viral vector.

23. The method of claim 20, wherein said vector is a non-viral vector.

24. The method of claim 17, wherein said cell is human.

25. A method of directing the differentiation of a HSC, a HPC, or a
combination thereof, to a cell of the myeloid lineage, said method
comprising introducing to said cell at least one biomarker selected from
the list consisting of Med8 (mediator of RNA polymerase II transcription,
subunit 8 homolog), Med14 (mediator complex subunit 14), GLIS2 (GLIS
family zinc finger 2), Tnfaip8l1 (tumor necrosis factor, alpha-induced
protein 8-like 1), and Mina (myc induced nuclear antigen).

26. The method of claim 25, wherein said biomarker is delivered to said
cell as a polypeptide.

27. The method of claim 25, wherein said biomarker is delivered to said
cell as a nucleic acid.

28. The method of claim 27, wherein said nucleic acid is contained within
a vector.

29. The method of claim 28, wherein said vector is a viral vector.

30. The method of claim 29, wherein said viral vector is selected from the
group consisting of a retroviral vector, an adenoviral vector, and an
adeno-associated viral vector.

31. The method of claim 28, wherein said vector is a non-viral vector.

32. The method of claim 25, wherein said cell is human.

Description:

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001]This is a National Stage application of PCT International
Application No. PCT/US2008/080324, filed Oct. 17, 2008, which in turn
claims the benefit pursuant to 35 U.S.C. §119(e) of U.S. Provisional
Application No. 60/999,595 filed on Oct. 19, 2007 which are hereby
incorporated by reference in their entirety herein.

BACKGROUND OF THE INVENTION

[0003]Hematopoiesis is defined by stepwise differentiation of
hematopoietic stem cells (HSC), through progenitor intermediates, to
terminally differentiated blood cells with vastly different morphologies
and functions. Regulation of this process is complex, involving
association of HSC with their specialized niche, signals from stromal
cells, epigenetic silencing of genetic programs specifying alternative
lineage fates, and crosstalk between HSC and non-stem cell neighbors. The
transcriptional control of HSC differentiation is still poorly
understood, despite advances in mouse genetic techniques that have
elucidated the role of certain pivotal molecules within the developmental
hierarchy. A few transcription factors have been shown to be essential
for specific lineages; for example, Early B-cell factor-1 (ebf1) in B
lymphocytes (Lin and Grosschedl, 1995, Nature 376:263-267), and gata2 in
megakaryocytic differentiation (Orkin et al., 1998, Stem Cells 16:79-83;
Ling et al., 2004, J. Exp. Med. 200:871-882). The roles of these and
other genes were identified empirically, and thus the number of genes
demonstrated to be critical for differentiation within each hematopoietic
population is extremely small or non-existent. The few global approaches
that have been used to study regulation of hematopoietic cells have
focused either on comparisons between HSC and other stem cell types
(Ivanova et al., 2002, Science 298:601-604; Ramalho et al., 2002,
Science, 298:597-600), or between HSC and pools of their differentiated
progeny (Toren et al., 2005, Stem Cells 23:1142-1153). The drawback of
these approaches is that many important regulatory candidates will not be
identified because of lack of purity in the comparator populations.

[0004]There exists in the art an urgent need to be able to direct
hematopoietic stem cell and hematopoietic progenitor cell differentiation
to a selected terminally differentiated blood cell. The present invention
answers this need.

SUMMARY OF THE INVENTION

[0005]One embodiment of the invention comprises a method of directing the
differentiation of a hematopoietic stem cell (HSC), a hematopoietic
progenitor cell (HPC), or a combination thereof, said method comprising
introducing into said cell a zinc finger protein (Zfp105), wherein said
Zfp105 upregulates expression of a natural killer (NK) cell-specific
polynucleotide in said cell, thereby inducing differentiation of said
cell into an NK cell. In one aspect of the invention, the Zfp105 is
delivered to said cell as a polypeptide. In another aspect of the
invention, the Zfp105 is delivered to said cell as a nucleic acid. In
another aspect of the invention, the nucleic acid is contained within a
vector. In another aspect of the invention, the vector is a viral vector.
In another aspect of the invention, the viral vector is selected from the
group consisting of a retroviral vector, an adenoviral vector, and an
adeno-associated viral vector. In another aspect of the invention, the
vector is a non-viral vector. In another aspect, the cell is a human
cell.

[0006]Another embodiment of the invention comprises a method of directing
the differentiation of a HSC, a HPC, or a combination thereof, said
method comprising introducing into said cell an Ets2, wherein said Ets2
upregulates expression of a monocyte-specific polynucleotide in said cell
thereby inducing differentiation of said cell into a monocyte. In one
aspect of the invention, the Ets2 is delivered to said cell as a
polypeptide. In another aspect of the invention, the Ets2 is delivered to
said cell as a nucleic acid. In another aspect of the invention, the
nucleic acid is contained within a vector. In another aspect of the
invention, the vector is a viral vector. In another aspect of the
invention, the viral vector is selected from the group consisting of a
retroviral vector, an adenoviral vector, and an adeno-associated viral
vector. In another aspect of the invention, the vector is a non-viral
vector. In another aspect, the cell is a human cell.

[0007]Yet another embodiment of the invention comprises a method of
directing the differentiation of a HSC, a HPC, or a combination thereof,
to a B-cell, said method comprising introducing to said cell at least one
biomarker selected from the list consisting of Chd7 (chromodomain
helicase DNA binding protein 7), Edaradd (EDAR (ectodysplasin-A
receptor)-associated death domain, 2210016F16Rik, Dzip1 (DAZ interacting
protein 1), and Tbl1x (transducin (beta)-like 1 X-linked). In one aspect
of the invention, the biomarker is delivered to said cell as a
polypeptide. In another aspect of the invention, the biomarker is
delivered to said cell as a nucleic acid. In another aspect of the
invention, the nucleic acid is contained within a vector. In another
aspect of the invention, the vector is a viral vector. In another aspect
of the invention, the viral vector is selected from the group consisting
of a retroviral vector, an adenoviral vector, and an adeno-associated
viral vector. In another aspect of the invention, the vector is a
non-viral vector. In another aspect, the cell is a human cell.

[0008]Still another embodiment of the invention comprises a method of
directing the differentiation of a HSC, a HPC, or a combination thereof,
to a cell of the myeloid lineage, said method comprising introducing to
said cell at least one biomarker selected from the list consisting of
Med8 (mediator of RNA polymerase II transcription, subunit 8 homolog),
Med14 (mediator complex subunit 14), GLIS2 (GLIS family zinc finger 2),
Tnfaip8l1 (tumor necrosis factor, alpha-induced protein 8-like 1), and
Mina (myc induced nuclear antigen). In one aspect of the invention, the
biomarker is delivered to said cell as a polypeptide. In another aspect
of the invention, the biomarker is delivered to said cell as a nucleic
acid. In another aspect of the invention, the nucleic acid is contained
within a vector. In another aspect of the invention, the vector is a
viral vector. In another aspect of the invention, the viral vector is
selected from the group consisting of a retroviral vector, an adenoviral
vector, and an adeno-associated viral vector. In another aspect of the
invention, the vector is a non-viral vector. In another aspect, the cell
is a human cell.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009]For the purpose of illustrating the invention, there are depicted in
the drawings certain embodiments of the invention. However, the invention
is not limited to the precise arrangements and instrumentalities of the
embodiments depicted in the drawings.

[0010]FIG. 1, comprising FIG. 1A and FIG. 1B, is a series of charts that
illustrate how global transcription profile analysis reveals
hematopoietic cell ontogeny. FIG. 1A depicts a dendrogram where the
right-most branch point indicates the cell types with the highest degree
of similarity. FIG. 1B depicts relative distance is collapsed to two
dimensions using PCA, the HSC resides at a midpoint between lymphocytes,
myeloid cells, and erythrocytes.

[0011]FIG. 2, comprising FIG. 2A through FIG. 2D, is a series of charts
depicting unique genetic fingerprints for hematopoietic cell types. FIG.
2A depicts three examples of genes expressed in a specific cell type. The
normalized (log2) expression intensity for each cell type for six example
genes is shown. The grey line indicates the threshold window, above which
a gene was considered to be expressed, and below which, non-expressed.
FIG. 2B depicts gene expression in all differentiated cells, lymphocytes,
or myeloid cells using a similar analysis. FIG. 2C is a heat map
summarizing finger print pattern. All the single cell type fingerprint
patterns are summarized in the heat map where the color intensity
indicates normalized level of expression (log2) with darker shades
corresponding to higher expression. FIG. 2D depicts the shared
fingerprint patterns.

[0012]FIG. 3, comprising FIG. 3A through FIG. 3C, is a series of charts
depicting examples of fingerprint genes and phenotype assessment. FIG. 3A
depicts selected genes from each of the cell-type-specific and shared
fingerprints in relation to their developmental relationships. FIG. 3B
depicts a Venn diagram which summarizes the number of shared genes in the
different fingerprints. FIG. 3C depicts a Venn diagram that summarizes
the number of knockout mice with hematopoietic phenotypes in each of the
indicated gene groups. For clarity, not all cell types and intersections
have been illustrated.

[0013]FIG. 4, comprising FIG. 4A through FIG. 4D, is a series of charts
which illustrate that T-cell and HSC activation share a similar
transcriptional repertoire. FIG. 4A depicts the results of a cluster
analysis. FIG. 4B depicts a pair-wise comparison between naive (bottom
panel) and activated T-cells (top panel, including both CD4+ and CD8+
cells) used to generate a list of genes up regulated in activated or
naive T-cells. FIG. 4C depicts shared fingerprints for CD4+, CD8+, and
`naive` and `activated T-cells. FIG. 4D depicts shared fingerprints for
CD4+, CD8+, and `naive` and `activated T-cells. Gene list sizes range
from 4 to 215, and are indicated to the right side of each heat map.

[0014]FIG. 5 is a graph which depicts the molecular pathway analysis of
hematopoietic cells using KEGG to identify which cell types express an
over- or under-abundance of components of a molecular pathways, with
significant differences between cell types determined by an ANOVA (one
way, a=0.05). For pathways containing genes found on the array, the mean
expression value for all genes within a pathway for each cell type is
displayed as a function of color density, where light coloration
corresponds to a low average expression and dark coloration to a high
average expression. Pathways are ordered by high expression within a cell
type from left to right. Below the heat map, the number of neutral to
over-abundant categories is denoted, and the percent of those pathways
that are signaling and metabolic is indicated. Myeloid cells (Mac. and
Grans.) show the greatest abundance of KEGG categories.

[0015]FIG. 6, comprising FIG. 6A through FIG. 6C, is a series of graphs
illustrating chromosomal expression density relative to chromatin state
for HSC compared to other cell types. FIG. 6A is a series of chromosomal
expression maps and then subtracted against each other. FIG. 6B is a
series of chromosomal expression maps, also examined along the
X-chromosome. FIG. 6C illustrates differences in expression density
between cell types on a per chromosome basis.

[0016]FIG. 7, comprising FIG. 7A through FIG. 7C, is a series of charts,
illustrating that retroviral transduction of fingerprint transcription
factors Ets2 and Zfp105 enforces lineage-specific cell fate. FIG. 7A
illustrates the experimental paradigm. FIG. 7B illustrates the overall
proportion of transduced cells (transduction, Tx.), and the proportions
of transduced myeloid, and lymphoid cells. FIG. 7C illustrates how Zfp105
transplants exhibited a depression in both B- and T-cells, and an overall
low level of apparent transduction.

[0017]FIG. 8 is a graph depicting the results of retroviral transduction
of Chd7 (chromodomain helicase DNA binding protein 7) in HSCs. The
percent of peripheral blood cells which undergo engraftment, are CD45.2,
B-, T- or myeloid cells is shown.

[0018]FIG. 9 is a graph depicting the results of retroviral transduction
of Edaradd (ectodysplasin-A receptor)-associated death domain) in HSCs.
The percent of peripheral blood cells which undergo engraftment, are
CD45.2, B-, T- or myeloid cells is shown.

[0019]FIG. 10 is a graph depicting the results of retroviral transduction
of Med8 (mediator of RNA polymerase II transcription, subunit 8 homolog)
in HSCs. The percent of peripheral blood cells which undergo engraftment,
are CD45.2, B-, T- or myeloid cells is shown.

[0020]FIG. 11 is a graph depicting the results of retroviral transduction
of Med14 (mediator complex subunit 14) in HSCs. The percent of peripheral
blood cells which undergo engraftment, are CD45.2, B-, T- or myeloid
cells is shown.

[0021]FIG. 12 is a graph depicting the results of retroviral transduction
of Glis2 (GLIS family zinc finger 2) in HSCs. The percent of peripheral
blood cells which undergo engraftment, are CD45.2, B-, T- or myeloid
cells is shown.

[0022]FIG. 13 is a graph depicting the results of retroviral transduction
of Dzip1 (DAZ interacting protein 1) in HSCs. The percent of peripheral
blood cells which undergo engraftment, are CD45.2, B-, T- or myeloid
cells is shown.

[0023]FIG. 14 is a graph depicting the results of retroviral transduction
of Tnfaip8l1 (tumor necrosis factor, alpha-induced protein 8-like 1) in
HSCs. The percent of peripheral blood cells which undergo engraftment,
are CD45.2, B-, T- or myeloid cells is shown.

[0024]FIG. 15 is a graph depicting the results of retroviral transduction
of 2210016F16Rik in HSCs. The percent of peripheral blood cells which
undergo engraftment, are CD45.2, B-, T- or myeloid cells is shown.

[0025]FIG. 16 is a graph depicting the results of retroviral transduction
of Tbl1x (transducin (beta)-like 1 X-linked) in HSCs. The percent of
peripheral blood cells which undergo engraftment, are CD45.2, B-, T- or
myeloid cells is shown.

[0026]FIG. 17 is a graph depicting the results of retroviral transduction
of Mina (myc induced nuclear antigen) in HSCs. The percent of peripheral
blood cells which undergo engraftment, are CD45.2, B-, T- or myeloid
cells is shown.

DETAILED DESCRIPTION OF THE INVENTION

[0027]The present invention is related to the discovery of biomarkers for
myeloid and lymphoid cell lineages. The present invention also discloses
biomarkers for differentiated cells, including Natural killer cells,
monocytes, B-cells and T-cells. The present invention is further related
to the discovery that overexpressing a biomarker in a hematopoietic stem
cell (HSC) or a hematopoietic progenitor cell (HPC) directs the
differentiation of the cell toward a specific cell lineage. Accordingly,
the invention encompasses a method of directing the differentiation of a
cell to a specific lineage by introducing a biomarker nucleic acid or a
biomarker protein into the cell.

Definitions:

[0028]Unless defined otherwise, all technical and scientific terms used
herein have the same meaning as commonly understood by one of ordinary
skill in the art to which the invention pertains. Although any methods
and materials similar or, equivalent to those described herein can be
used in the practice for testing of the present invention, the preferred
materials and methods are described herein. In describing and claiming
the present invention, the following terminology will be used.

[0029]It is also to be understood that the terminology used herein is for
the purpose of describing particular embodiments only, and is not
intended to be limiting.

[0030]As used in this specification and the appended claims, the singular
forms "a", "an", and "the" include plural referents unless the content
clearly dictates otherwise. Thus, for example, reference to "a protein"
includes a combination of two or more proteins, and the like.

[0031]"About" as used herein when referring to a measurable value such as
an amount, a temporal duration, and the like, is meant to encompass
variations of ±20% or ±10%, more preferably ±5%, even more
preferably ±1%, and still more preferably ±0.1% from the specified
value, as such variations are appropriate to perform the disclosed
methods.

[0032]A "biomarker" is any gene, protein, or metabolite whose level of
expression in a tissue, cell or bodily fluid is unique to that tissue,
cell, or bodily fluid.

[0033]The phrase "body sample" as used herein, is intended to include any
sample comprising a cell, a tissue, or a bodily fluid in which expression
of a biomarker can be detected. Examples of such body samples include but
are not limited to blood, lymph, biopsies and smears. Samples that are
liquid in nature are referred to herein as "bodily fluids." Body samples
may be obtained from a patient by a variety of techniques including, for
example, by scraping or swabbing an area or by using a needle to aspirate
bodily fluids. Methods for collecting various body samples are well known
in the art.

[0034]A "coding region" of a gene consists of the nucleotide residues of
the coding strand of the gene and the nucleotides of the non-coding
strand of the gene which are homologous with or complementary to,
respectively, the coding region of an mRNA molecule which is produced by
transcription of the gene.

[0035]A "coding region" of an mRNA molecule also consists of the
nucleotide residues of the mRNA molecule which are matched with an
anti-codon region of a transfer RNA molecule during translation of the
mRNA molecule or which encode a stop codon. The coding region may thus
include nucleotide residues corresponding to amino acid residues which
are not present in the mature protein encoded by the mRNA molecule (e.g.,
amino acid residues in a protein export signal sequence).

[0036]"Complementary" as used herein to refer to a nucleic acid, refers to
the broad concept of sequence complementarity between regions of two
nucleic acid strands or between two regions of the same nucleic acid
strand. It is known that an adenine residue of a first nucleic acid
region is capable of forming specific hydrogen bonds ("base pairing")
with a residue of a second nucleic acid region which is antiparallel to
the first region if the residue is thymine or uracil. Similarly, it is
known that a cytosine residue of a first nucleic acid strand is capable
of base pairing with a residue of a second nucleic acid strand which is
antiparallel to the first strand if the residue is guanine. A first
region of a nucleic acid is complementary to a second region of the same
or a different nucleic acid if, when the two regions are arranged in an
antiparallel fashion, at least one nucleotide residue of the first region
is capable of base pairing with a residue of the second region.
Preferably, the first region comprises a first portion and the second
region comprises a second portion, whereby, when the first and second
portions are arranged in an antiparallel fashion, at least about 50%, and
preferably at least about 75%, at least about 90%, or at least about 95%
of the nucleotide residues of the first portion are capable of base
pairing with nucleotide residues in the second portion. More preferably,
all nucleotide residues of the first portion are capable of base pairing
with nucleotide residues in the second portion.

[0037]The term "DNA" as used herein is defined as deoxyribonucleic acid.

[0038]The term "dysregulation" as used herein, describes an abnormality of
the cell content of blood. The abnormality may stem from a traumatic
event (including hemorrhage) or a disease process (including HIV or
cancer). The dysregulation may be detected by a complete blood count
(CBC) wherein there is either depletion or over production of a
particular blood cell type detected in a blood sample obtained from an
individual as compared to a blood sample obtained from one or more normal
individuals, or from the same individual at a different time point.
Examples of such a dysregulation include elevated red blood cells, low
numbers of red blood cells, elevated lymphocytes (leukocytosis) or low
numbers of lymphocytes (leucopenia).

[0039]"Encoding" refers to the inherent property of specific sequences of
nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to
serve as templates for synthesis of other polymers and macromolecules in
biological processes having either a defined sequence of nucleotides
(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the
biological properties resulting there from. Thus, a gene encodes a
protein if transcription and translation of mRNA corresponding to that
gene produces the protein in a cell or other biological system. Both the
coding strand, the nucleotide sequence of which is identical to the mRNA
sequence and is usually provided in sequence listings, and the non-coding
strand, used as the template for transcription of a gene or cDNA, can be
referred to as encoding the protein or other product of that gene or
cDNA.

[0040]Unless otherwise specified, a "nucleotide sequence encoding an amino
acid sequence" includes all nucleotide sequences that are degenerate
versions of each other and that encode the same amino acid sequence.
Nucleotide sequences that encode proteins and RNA may include introns.

[0041]"Isolated" means altered or removed from the natural state. For
example, a nucleic acid or a peptide naturally present in a living animal
is not "isolated," but the same nucleic acid or peptide partially or
completely separated from the coexisting materials of its natural state
is "isolated." An isolated nucleic acid or protein can exist in
substantially purified form, or can exist in a non-native environment
such as, for example, a host cell.

[0042]An "isolated nucleic acid" refers to a nucleic acid segment or
fragment which has been separated from sequences which flank it in a
naturally occurring state, i.e., a DNA fragment which has been removed
from the sequences which are normally adjacent to the fragment, i.e., the
sequences adjacent to the fragment in a genome in which it naturally
occurs. The term also applies to nucleic acids which have been
substantially purified from other components which naturally accompany
the nucleic acid, i.e., RNA or DNA or proteins, which naturally accompany
it in the cell. The term therefore includes, for example, a recombinant
DNA which is incorporated into a vector, into an autonomously replicating
plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote,
or which exists as a separate molecule (i.e., as a cDNA or a genomic or
cDNA fragment produced by PCR or restriction enzyme digestion)
independent of other sequences. It also includes a recombinant DNA which
is part of a hybrid gene encoding additional polypeptide sequence.

[0043]In the context of the present invention, the following abbreviations
for the commonly occurring nucleic acid bases are used. "A" refers to
adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to
thymidine, and "U" refers to uridine.

[0044]Unless otherwise specified, a "nucleotide sequence encoding an amino
acid sequence" includes all nucleotide sequences that are degenerate
versions of each other and that encode the same amino acid sequence. The
phrase nucleotide sequence that encodes a protein or an RNA may also
include introns to the extent that the nucleotide sequence encoding the
protein may in some version contain an intron(s).

[0045]The term "polynucleotide" as used herein is defined as a chain of
nucleotides. Furthermore, nucleic acids are polymers of nucleotides.
Thus, nucleic acids and polynucleotides as used herein are
interchangeable. One skilled in the art has the general knowledge that
nucleic acids are polynucleotides, which can be hydrolyzed into the
monomeric "nucleotides." The monomeric nucleotides can be hydrolyzed into
nucleosides. As used herein polynucleotides include, but are not limited
to, all nucleic acid sequences which are obtained by any means available
in the art, including, without limitation, recombinant means, i.e., the
cloning of nucleic acid sequences from a recombinant library or a cell
genome, using ordinary cloning technology and PCR®, and the like, and
by synthetic means.

[0046]As used herein, the terms "peptide," "polypeptide," and "protein"
are used interchangeably, and refer to a compound comprised of amino acid
residues covalently linked by peptide bonds. A protein or peptide must
contain at least two amino acids, and no limitation is placed on the
maximum number of amino acids that can comprise a protein's or peptide's
sequence. Polypeptides include any peptide or protein comprising two or
more amino acids joined to each other by peptide bonds. As used herein,
the term refers to both short chains, which also commonly are referred to
in the art as peptides, oligopeptides and oligomers, for example, and to
longer chains, which generally are referred to in the art as proteins, of
which there are many types. "Polypeptides" include, for example,
biologically active fragments, substantially homologous polypeptides,
oligopeptides, homodimers, heterodimers, variants of polypeptides,
modified polypeptides, derivatives, analogs, fusion proteins, among
others. The polypeptides include natural peptides, recombinant peptides,
synthetic peptides, or a combination thereof.

[0047]The term "progeny" as used herein refers to a descendent or
offspring.

[0048]In one use, the term progeny refers to a clonal descendent which is
genetically identical to the parent. In another usage, the term progeny
refers to cells which have differentiated from the parent cell.

[0049]The term "RNA" as used herein is defined as ribonucleic acid.

[0050]The term "recombinant DNA" as used herein is defined as DNA produced
by joining pieces of DNA from different sources.

[0051]The term "recombinant polypeptide" as used herein is defined as a
polypeptide produced by using recombinant DNA methods.

[0052]By the term "specifically binds," as used herein, is meant a
molecule, such as an antibody, which recognizes and binds to a cell
surface molecule or feature, but does not substantially recognize or bind
other molecules or features in a sample.

[0053]"Stromal cells" as used herein comprise fibroblasts and mesenchymal
cells, with or without other cells and elements, and can be seeded prior
to, or substantially at the same time as the hematopoietic progenitor
cells, therefore establishing conditions that favor the subsequent
attachment and growth of hematopoietic progenitor cells. Fibroblasts can
be obtained via a biopsy from any tissue or organ, and include fetal
fibroblasts. These fibroblasts and mesenchymal cells may be transfected
with exogenous DNA that encodes, for example, one of the hematopoietic
growth factors described above. Stromal cells can be of lymphoid or
non-lymphoid origin.

[0054]"Stromal cell conditioned medium" refers to medium in which the
aforementioned stromal cells have been incubated. The incubation is
performed for a period sufficient to allow the stromal cells to secrete
factors into the medium. Such "stromal cell conditioned medium" can then
be used to supplement the culture of hematopoietic cells promoting their
proliferation and/or differentiation.

[0055]As used herein, "conjugated" refers to covalent attachment of one
molecule to a second molecule.

[0056]"Variant" as the term is used herein, is a nucleic acid sequence or
a peptide sequence that differs in sequence from a reference nucleic acid
sequence or peptide sequence respectively, but retains essential
properties of the reference molecule. Changes in the sequence of a
nucleic acid variant may not alter the amino acid sequence of a peptide
encoded by the reference nucleic acid, or may result in amino acid
substitutions, additions, deletions, fusions and truncations. Changes in
the sequence of peptide variants are typically limited or conservative,
so that the sequences of the reference peptide and the variant are
closely similar overall and, in many regions, identical. A variant and
reference peptide can differ in amino acid sequence by one or more
substitutions, additions, deletions in any combination. A variant of a
nucleic acid or peptide can be a naturally occurring such as an allelic
variant, or can be a variant that is not known to occur naturally.
Non-naturally occurring variants of nucleic acids and peptides may be
made by mutagenesis techniques or by direct synthesis.

Description:

[0057]The present invention identifies, for the first time, a number of
biomarkers which are specific for the myeloid lineage or the lymphoid
lineage. The present invention also identifies biomarkers for
differentiated cells, including NK cells, monocytes, B-cells, T-cells and
monocytes.

[0058]The present invention springs from the discovery that hematopoiesis
can be directed toward a specific cell lineage by expressing a specific
biomarker nucleic acid or protein in a cell. As such, The present
invention includes methods of directing the differentiation of a cell
toward the myloid or lymphoid cell lineages by expressing these
biomarkers in the cell.

I. Compositions

Hematopoietic Cells

[0059]A cell of the invention can be a pluripotent hematopoietic stem cell
(HSC) or a multipotent hematopoietic progenitor cell (HPC). Hematopoietic
stem cells and hematopoietic progenitor cells are immature blood cells
with the capacity to self-renew and to differentiate into more mature
blood cells. Pluripotent HSCs are capable of differentiating into any
blood cell type. Multipotent HPCs are capable into differentiating into a
blood cell of a particular lineage. HPCs may be either a common myeloid
progenitor cell or a common lymphoid progenitor cell. The common myeloid
progenitor cell gives rise to cells of the myeloid lineage and the common
lymphoid progenitor cell gives rise to cells of the lymphoid lineage.
Cells derived from a common myeloid progenitor cell include granulocytes
(e.g., neutrophils, eosinophils, basophils), erythrocytes (e.g.,
reticulocytes, erythrocytes), thrombocytes (e.g., megakaryoblasts,
platelet producing megakaryocytes, platelets), and monocytes (e.g.
monocytes, macrophages). Cells derived from a common lymphoid progenitor
cell include B-cells, T-cells, and natural killer (NK) cells.

[0061]In certain embodiments, a cell of the invention is autologous (e.g.,
originate from the same individual). In some embodiments, a cell of the
invention is non-autologous. A cell of the present invention may be
allogenic, syngenic or xenogenic. "Allogeneic," as used herein, refers to
cells of the same species that differ genetically to the cell in
comparison. "Syngeneic," as used herein, refers to cells of a different
subject that are genetically identical to the cell in comparison.
"Xenogeneic," as used herein, refers to cells of a different species to
the cell in comparison. In one embodiment of the invention, the cells of
the invention are of human origin.

Biomarkers of Hematopoietic Lineage

[0062]In one embodiment of the invention, a biomarker of the invention
comprises any gene, nucleic acid, protein, peptide or fragment thereof
that is specifically expressed by cells of the myeloid lineage, but not
by cells of the lymphoid lineage. In another embodiment of the invention,
a biomarker of the invention comprises any gene, nucleic acid, protein,
peptide or fragment thereof that is expressed by cells of the lymphoid
lineage, but not in cells of the myeloid lineage. Biomarkers of
particular interest include genes and proteins involved in hematopoiesis.
In some embodiments, the biomarker is an enzyme, a protein associated
with cellular differentiation, or a transcription factor. In another
embodiment of the invention, expression of a biomarker in a cell of the
invention directs the differentiation of that cell to a specific cell
lineage.

[0063]"Biomarker nucleic acids," as used herein, include DNA comprising
the entire or partial sequence of the nucleic acid sequence encoding a
biomarker, or the complement of such a sequence. Biomarker nucleic acids
useful in the invention should be considered to include both DNA and RNA
comprising the entire or partial sequence of any of the nucleic acid
sequences of interest.

[0064]"Biomarker proteins", as used herein, should be considered to
comprise the entire or partial amino acid sequence of any of the
biomarker proteins or polypeptides.

[0066]Ets2 is a member of the ETS family of transcription factors, many of
which have been implicated in tumor progression. Ets2 is implicated in
acute myeloid leukemia and has been shown to play a role in inhibiting
apoptosis of macrophages (Baldus et al., 2004, Proc. Natl. Acad. Sci.
U.S.A. 101:3915-1920; Sevilla et al., 1999, Mol. Cell. Biol.
19:2624-2634).

[0068]Chd7 (chromodomain helicase DNA binding protein 7), a B-cell
fingerprint gene, is a putative transcription factor associated with
CHARGE syndrome in humans. CHARGE is a complex human disorder with such
clinical features as coloboma of the eye, choanal atresia or stenosis,
abnormalities of the cranial nerves, otological deformities, and defects
of one or more major organ systems. At least one gene has been identified
with CHARGE. Heterozygous Chd7 knock-out mice display the symptoms of
CHARGE syndrome.

[0069]Edaradd (EDAR (ectodysplasin-A receptor)-associated death domain), a
B-cell fingerprint gene, is a cytoplasmic receptor previously implicated
in fertility and hair follicle development in mice.

[0070]Med8 (mediator of RNA polymerase II transcription, subunit 8
homolog), a Differentiated fingerprint gene, skews differentiation toward
the myeloid lineages. Myeloid skewing is consistent with a role in stem
cell expansion. Med8 is a putative transcription factor that may be part
of the mediator complex.

[0071]Med14 (mediator complex subunit 14), an HSC fingerprint gene, is a
putative transcription factor that may be part of the mediator complex.

[0079]The present invention also provides for analogs of polypeptides
which comprise a biomarker protein. Analogs may differ from naturally
occurring proteins or polypeptides by conservative amino acid sequence
differences or by modifications which do not affect sequence, or by both.
For example, conservative amino acid changes may be made, which although
they alter the primary sequence of the protein or polypeptide, do not
normally alter its function (e.g., secretion and capable of blocking
virus infection). Conservative amino acid substitutions typically include
substitutions within the following groups: [0080]glycine, alanine;
[0081]valine, isoleucine, leucine; [0082]aspartic acid, glutamic acid;
[0083]asparagine, glutamine; [0084]serine, threonine; [0085]lysine,
arginine; [0086]phenylalanine, tyrosine.

[0087]Modifications (which do not normally alter primary sequence) include
in vivo, or in vitro, chemical derivatization of polypeptides, e.g.,
acetylation, or carboxylation. Also included are modifications of
glycosylation, e.g., those made by modifying the glycosylation patterns
of a polypeptide during its synthesis and processing or in further
processing steps; e.g., by exposing the polypeptide to enzymes which
affect glycosylation, e.g., mammalian glycosylating or deglycosylating
enzymes. Also embraced are sequences which have phosphorylated amino acid
residues, e.g., phosphotyrosine, phosphoserine, or phosphothreonine.

[0088]The present invention should also be construed to encompass
"mutants," "derivatives," and "variants" of the biomarker proteins of the
invention (or of the DNA encoding the same) which mutants, derivatives
and variants are altered in one or more amino acids (or, when referring
to the nucleotide sequence encoding the same, are altered in one or more
base pairs) such that the resulting peptide (or DNA) is not identical to
the sequences recited herein, but has the same biological property as the
biomarker proteins disclosed herein, in that the proteins have
biological/biochemical properties. A biological property of the
polypeptides of the present invention should be construed but not be
limited to include, the ability to mediate normal hematopoiesis.

[0089]Further, the invention should be construed to include naturally
occurring variants or recombinantly derived mutants of biomarker proteins
sequences, which variants or mutants render the polypeptide encoded
thereby either more, less, or just as biologically active as wild type
biomarker proteins.

[0090]The present invention should not be construed to be limited to the
biomarkers nucleic acids and biomarker proteins recited herein, but
rather should be construed to encompass any biomarker, which, when
expressed in a cell of the invention directs the differentiation of said
cell toward a selected cell lineage. Methods for identifying such
biomarkers are within the skill of the art, and are described herein.

Biomarker Nucleic Acids: Synthesis

[0091]Any number of procedures may be used for the generation of an
isolated biomarker nucleic acid as well as derivative or variant forms of
an isolated biomarker nucleic acid, using recombinant DNA methodology
well known in the art (see Sambrook et al., 2001, Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, New York; Ausubel
et al., 2001, Current Protocols in Molecular Biology, Green & Wiley, New
York) and by direct synthesis. For recombinant and in vitro
transcription, DNA encoding RNA molecules can be obtained from known
clones, by synthesizing a DNA molecule encoding an RNA molecule, or by
cloning the gene encoding the RNA molecule. Techniques for in vitro
transcription of RNA molecules and methods for cloning genes encoding
known RNA molecules are described by, for example, Sambrook et al.

[0092]An isolated biomarker nucleic acid of the present invention can be
produced using conventional nucleic acid synthesis or by recombinant
nucleic acid methods known in the art and described elsewhere herein
(2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, New York) and Ausubel et al. (2001, Current Protocols
in Molecular Biology, Green & Wiley, New York).

[0093]As an example, a method for synthesizing nucleic acids de novo
involves the organic synthesis of a nucleic acid from nucleoside
derivatives. This synthesis may be performed in solution or on a solid
support. One type of organic synthesis is the phosphotriester method,
which has been used to prepare gene fragments or short genes. In the
phosphotriester method, oligonucleotides are prepared which can then be
joined together to form longer nucleic acids. For a description of this
method, see Narang, et al., (1979, Meth. Enzymol., 68: 90) and U.S. Pat.
No. 4,356,270. The phosphotriester method can be used in the present
invention to synthesize an isolated snRNA.

[0094]In addition, the compositions of the present invention can be
synthesized in whole or in part, or an isolated biomarker nucleic acid
can be conjugated to another nucleic acid using organic synthesis such as
the phosphodiester method, which has been used to prepare a tRNA gene.
See Brown, et al. (1979, Meth. Enzymol., 68: 109) for a description of
this method. As in the phosphotriester method, the phosphodiester method
involves synthesis of oligonucleotides which are subsequently joined
together to form the desired nucleic acid.

[0095]A third method for synthesizing nucleic acids, described in U.S.
Pat. No. 4,293,652, is a hybrid of the above-described organic synthesis
and molecular cloning methods. In this process, the appropriate number of
oligonucleotides to make up the desired nucleic acid sequence is
organically synthesized and inserted sequentially into a vector which is
amplified by growth prior to each succeeding insertion.

[0096]In addition, molecular biological methods, such as using a nucleic
acid as a template for a PCR or LCR reaction, or cloning a nucleic acid
into a vector and transforming a cell with the vector can be used to make
large amounts of the nucleic acid of the present invention.

[0097]Any of a variety of procedures may be used to molecularly clone
biomarker cDNA. These methods include, but are not limited to, direct
functional expression of a biomarker gene, including but not limited to
Ets2, Zfp105, Chd7, EDAR, Med8, Med14, Glis2, Dzip1, Tnfaip8l1,
2210016F16Rik, Tbl1x, and Mina, following the construction of a
biomarker-containing cDNA library in an appropriate expression vector
system.

[0098]It is readily apparent to those skilled in the art that suitable
cDNA libraries may be prepared from cells or cell lines which express a
biomarker of interest. By way of a non-limiting example, cells or
cell-lines which express Ets2 would be useful in the preparation of a
cDNA library. Similarly, cells or cell lines which express Zfp105 would
be useful in the preparation of a cDNA library.

[0099]Preparation of cDNA libraries can be performed by standard
techniques well known in the art. Well known cDNA library construction
techniques can be found for example, in Maniatis, T., Fritsch, E. F.,
Sambrook, J., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y., 1982).

[0100]It is also readily apparent to those skilled in the art that DNA
encoding a biomarker may also be isolated from a suitable genomic DNA
library. Construction of genomic DNA libraries can be performed by
standard techniques well known in the art. Well known genomic DNA library
construction techniques can be found in Maniatis, T., Fritsch, E. F.,
Sambrook, J. in Molecular Cloning: A Laboratory Manuel (Cold Spring
Harbor Laboratory, Cold Spring Harbor, N.Y., 1982).

[0101]Biomarker molecules may also be obtained by recombinantly
engineering them from DNA encoding the partial or complete amino acid
sequence of a biomarker. Using recombinant DNA techniques, DNA molecules
are constructed which encode at least a portion of a biomarker molecule
such at Ets2, Zfp105, Chd7, EDAR, Med8, Med14, Glis2, Dzip1, Tnfaip8l1,
2210016F16Rik, Tbl1x, and Mina. Standard recombinant DNA techniques are
used such as those found in Maniatis, et al., supra.

[0102]The cloned biomarker cDNA obtained through the methods described
above may be recombinantly expressed by molecular cloning into an
expression vector containing a suitable promoter and other appropriate
transcription regulatory elements, and transferred into prokaryotic or
eukaryotic host cells to produce recombinant biomarker. Techniques for
such manipulations are fully described in Maniatis, T, et al., supra, and
are well known in the art.

[0103]In other related aspects, the invention includes an isolated nucleic
acid encoding a biomarker, operably linked to a nucleic acid comprising a
promoter/regulatory sequence such that the nucleic acid is preferably
capable of directing expression of the biomarker protein encoded by the
nucleic acid. Thus, the invention encompasses expression vectors and
methods for the introduction of exogenous DNA into cells with concomitant
expression of the exogenous DNA in the cells such as those described, for
example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Laboratory, New York), and in Ausubel et al.
(1997, Current Protocols in Molecular Biology, John Wiley & Sons, New
York). The incorporation of a desired polynucleotide into a vector and
the choice of vectors is well-known in the art as described in, for
example, Sambrook et al., supra, and Ausubel et al., supra.

[0104]The biomarker polynucleotide can be cloned into a number of types of
vectors. However, the present invention should not be construed to be
limited to any particular vector. Instead, the present invention should
be construed to encompass a wide plethora of vectors which are readily
available and/or well-known in the art. For example, a biomarker
polynucleotide of the invention can be cloned into a vector including,
but not limited to a plasmid, a phagemid, a phage derivative, an animal
viruse, and a cosmid. Vectors of particular interest include expression
vectors, replication vectors, probe generation vectors, and sequencing
vectors.

[0105]In specific embodiments, the expression vector is selected from the
group consisting of a viral vector, a bacterial vector and a mammalian
cell vector. Numerous expression vector systems exist that comprise at
least a part or all of the compositions discussed above. Prokaryote-
and/or eukaryote-vector based systems can be employed for use with the
present invention to produce polynucleotides, or their cognate
polypeptides. Many such systems are commercially and widely available.

[0106]Further, the expression vector may be provided to a cell in the form
of a viral vector. Viral vector technology is well known in the art and
is described, for example, in Sambrook et al. (2001), and in Ausubel et
al. (1997), and in other virology and molecular biology manuals. Viruses,
which are useful as vectors include, but are not limited to,
retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and
lentiviruses. In general, a suitable vector contains an origin of
replication functional in at least one organism, a promoter sequence,
convenient restriction endonuclease sites, and one or more selectable
markers. (See, e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No.
6,326,193.

[0107]At least one module in each promoter functions to position the start
site for RNA synthesis. The best known example of this is the TATA box,
but in some promoters lacking a TATA box, such as the promoter for the
mammalian terminal deoxynucleotidyl transferase gene and the promoter for
the SV40 genes, a discrete element overlying the start site itself helps
to fix the place of initiation.

[0108]Additional promoter elements, i.e., enhancers, regulate the
frequency of transcriptional initiation. Typically, these are located in
the region 30-110 bp upstream of the start site, although a number of
promoters have recently been shown to contain functional elements
downstream of the start site as well. The spacing between promoter
elements frequently is flexible, so that promoter function is preserved
when elements are inverted or moved relative to one another. In the
thymidine kinase (tk) promoter, the spacing between promoter elements can
be increased to 50 bp apart before activity begins to decline. Depending
on the promoter, it appears that individual elements can function either
co-operatively or independently to activate transcription.

[0109]A promoter may be one naturally associated with a gene or
polynucleotide sequence, as may be obtained by isolating the 5'
non-coding sequences located upstream of the coding segment and/or exon.
Such a promoter can be referred to as "endogenous." Similarly, an
enhancer may be one naturally associated with a polynucleotide sequence,
located either downstream or upstream of that sequence. Alternatively,
certain advantages will be gained by positioning the coding
polynucleotide segment under the control of a recombinant or heterologous
promoter, which refers to a promoter that is not normally associated with
a polynucleotide sequence in its natural environment. A recombinant or
heterologous enhancer refers also to an enhancer not normally associated
with a polynucleotide sequence in its natural environment. Such promoters
or enhancers may include promoters or enhancers of other genes, and
promoters or enhancers isolated from any other prokaryotic, viral, or
eukaryotic cell, and promoters or enhancers not "naturally occurring,"
i.e., containing different elements of different transcriptional
regulatory regions, and/or mutations that alter expression. In addition
to producing nucleic acid sequences of promoters and enhancers
synthetically, sequences may be produced using recombinant cloning and/or
nucleic acid amplification technology, including PCR®, in connection
with the compositions disclosed herein (U.S. Pat. No. 4,683,202, U.S.
Pat. No. 5,928,906). Furthermore, it is contemplated the control
sequences that direct transcription and/or expression of sequences within
non-nuclear organelles such as mitochondria, chloroplasts, and the like,
can be employed as well.

[0110]Naturally, it will be important to employ a promoter and/or enhancer
that effectively directs the expression of the DNA segment in the cell
type, organelle, and organism chosen for expression. Those of skill in
the art of molecular biology generally know how to use promoters,
enhancers, and cell type combinations for protein expression, for
example, see Sambrook et al. (2001). The promoters employed may be
constitutive, tissue-specific, inducible, and/or useful under the
appropriate conditions to direct high level expression of the introduced
DNA segment, such as is advantageous in the large-scale production of
recombinant proteins and/or peptides. The promoter may be heterologous or
endogenous.

[0111]A promoter sequence exemplified in the experimental examples
presented herein is the immediate early cytomegalovirus (CMV) promoter
sequence. This promoter sequence is a strong constitutive promoter
sequence capable of driving high levels of expression of any
polynucleotide sequence operatively linked thereto. However, other
constitutive promoter sequences may also be used, including, but not
limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor
virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat
(LTR) promoter, Moloney virus promoter, the avian leukemia virus
promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus
promoter, as well as human gene promoters such as, but not limited to,
the actin promoter, the myosin promoter, the hemoglobin promoter, and the
muscle creatine promoter. Further, the invention should not be limited to
the use of constitutive promoters. Inducible promoters are also
contemplated as part of the invention. The use of an inducible promoter
in the invention provides a molecular switch capable of turning on
expression of the polynucleotide sequence which it is operatively linked
when such expression is desired, or turning off the expression when
expression is not desired. Examples of inducible promoters include, but
are not limited to a metallothionine promoter, a glucocorticoid promoter,
a progesterone promoter, and a tetracycline promoter. Further, the
invention includes the use of a tissue specific promoter, which promoter
is active only in a desired tissue. Tissue specific promoters are well
known in the art and include, but are not limited to, the HER-2 promoter
and the PSA associated promoter sequences.

[0112]In order to assess the expression of the biomarker, the expression
vector to be introduced into a cell can also contain either a selectable
marker gene or a reporter gene or both to facilitate identification and
selection of expressing cells from the population of cells sought to be
transfected or infected through viral vectors. In other embodiments, the
selectable marker may be carried on a separate piece of DNA and used in a
co-transfection procedure. Both selectable markers and reporter genes may
be flanked with appropriate regulatory sequences to enable expression in
the host cells. Useful selectable markers are known in the art and
include, for example, antibiotic-resistance genes, such as neo and the
like.

[0113]Reporter genes are used for identifying potentially transfected
cells and for evaluating the functionality of regulatory sequences.
Reporter genes that encode for easily assayable proteins are well known
in the art. In general, a reporter gene is a gene that is not present in
or expressed by the recipient organism or tissue and that encodes a
protein whose expression is manifested by some easily detectable
property, e.g., enzymatic activity. Expression of the reporter gene is
assayed at a suitable time after the DNA has been introduced into the
recipient cells.

[0114]Suitable reporter genes may include genes encoding luciferase,
beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline
phosphatase, or the green fluorescent protein gene (see, e.g., Ui-Tei et
al., 2000 FEBS Lett. 479:79-82). Suitable expression systems are well
known and may be prepared using well known techniques or obtained
commercially. Internal deletion constructs may be generated using unique
internal restriction sites or by partial digestion of non-unique
restriction sites. Constructs may then be transfected into cells that
display high levels of siRNA polynucleotide and/or polypeptide
expression. In general, the construct with the minimal 5' flanking region
showing the highest level of expression of reporter gene is identified as
the promoter. Such promoter regions may be linked to a reporter gene and
used to evaluate agents for the ability to modulate promoter-driven
transcription.

Biomarker Proteins: Synthesis

[0115]Biomarker proteins useful in the invention, including, but not
limited to, Ets2, Zfp105, Chd7, EDAR, Med8, Med14, Glis2, Dzip1,
Tnfaip8l1, 2210016F16Rik, Tbl1x, and Mina, may be obtained using standard
methods known to the skilled artisan. Such methods include chemical
organic synthesis or biological means. Biological means include
purification from a biological source, recombinant synthesis and in vitro
translation systems, using methods well known in the art.

[0116]A peptide may be chemically synthesized by Merrifield-type solid
phase peptide synthesis. This method may be routinely performed to yield
peptides up to about 60-70 residues in length, and may, in some cases, be
utilized to make peptides up to about 100 amino acids long. Larger
peptides may also be generated synthetically via fragment condensation or
native chemical ligation (Dawson et al., 2000, Ann. Rev. Biochem.
69:923-960). An advantage to the utilization of a synthetic peptide route
is the ability to produce large amounts of peptides, even those that
rarely occur naturally, with relatively high purities, i.e., purities
sufficient for research, diagnostic or therapeutic purposes.

[0117]Solid phase peptide synthesis is described by Stewart et al. in
Solid Phase Peptide Synthesis, 2nd Edition, 1984, Pierce Chemical
Company, Rockford, Ill.; and Bodanszky and Bodanszky in The Practice of
Peptide Synthesis, 1984, Springer-Verlag, New York. At the outset, a
suitably protected amino acid residue is attached through its carboxyl
group to a derivatized, insoluble polymeric support, such as cross-linked
polystyrene or polyamide resin. "Suitably protected" refers to the
presence of protecting groups on both the α-amino group of the
amino acid, and on any side chain functional groups. Side chain
protecting groups are generally stable to the solvents, reagents and
reaction conditions used throughout the synthesis, and are removable
under conditions which will not affect the final peptide product.
Stepwise synthesis of the oligopeptide is carried out by the removal of
the N-protecting group from the initial amino acid, and coupling thereto
of the carboxyl end of the next amino acid in the sequence of the desired
peptide. This amino acid is also suitably protected. The carboxyl of the
incoming amino acid can be activated to react with the N-terminus of the
support-bound amino acid by formation into a reactive group, such as
formation into a carbodiimide, a symmetric acid anhydride, or an "active
ester" group, such as hydroxybenzotriazole or pentafluorophenyl esters.

[0118]Examples of solid phase peptide synthesis methods include the BOC
method, which utilizes tert-butyloxcarbonyl as the α-amino
protecting group, and the FMOC method, which utilizes
9-fluorenylmethyloxcarbonyl to protect the α-amino of the amino
acid residues. Both methods are well-known by those of skill in the art.

[0119]Incorporation of N- and/or C-blocking groups may also be achieved
using protocols conventional to solid phase peptide synthesis methods.
For incorporation of C-terminal blocking groups, for example, synthesis
of the desired peptide is typically performed using, as solid phase, a
supporting resin that has been chemically modified so that cleavage from
the resin results in a peptide having the desired C-terminal blocking
group. To provide peptides in which the C-terminus bears a primary amino
blocking group, for instance, synthesis is performed using a
p-methylbenzhydrylamine (MBHA) resin, so that, when peptide synthesis is
completed, treatment with hydrofluoric acid releases the desired
C-terminally amidated peptide. Similarly, incorporation of an
N-methylamine blocking group at the C-terminus is achieved using
N-methylaminoethyl-derivatized DVB (divinylbenzene), resin, which upon
hydrofluoric acid (HF) treatment releases a peptide bearing an
N-methylamidated C-terminus. Blockage of the C-terminus by esterification
can also be achieved using conventional procedures. This entails use of
resin/blocking group combination that permits release of side-chain
peptide from the resin, to allow for subsequent reaction with the desired
alcohol, to form the ester function. FMOC protecting group, in
combination with DVB resin derivatized with methoxyalkoxybenzyl alcohol
or equivalent linker, can be used for this purpose, with cleavage from
the support being affected by trifluoroacetic acid (TFA) in
dicholoromethane. Esterification of the suitably activated carboxyl
function, e.g. with dicyclohexylcarbodiimide (DCC), can then proceed by
addition of the desired alcohol, followed by de-protection and isolation
of the esterified peptide product.

[0120]Incorporation of N-terminal blocking groups may be achieved while
the synthesized peptide is still attached to the resin, for instance by
treatment with a suitable anhydride and nitrile. To incorporate an acetyl
blocking group at the N-terminus, for instance, the resin-coupled peptide
can be treated with 20% acetic anhydride in acetonitrile. The N-blocked
peptide product may then be cleaved from the resin, de-protected and
subsequently isolated.

[0121]Prior to its use in accordance with the invention, a biomarker
protein, peptide, or fragment thereof is purified to remove contaminants.
Any one of a number of a conventional purification procedures may be used
to attain the required level of purity including, for example,
reversed-phase high-pressure liquid chromatoraphy (HPLC) using an
alkylated silica column such as C4-, C8- or C18-silica. A
gradient mobile phase of increasing organic content is generally used to
achieve purification, for example, acetonitrile in an aqueous buffer,
usually containing a small amount of trifluoroacetic acid. Ion-exchange
chromatography can be also used to separate polypeptides based on their
charge. Affinity chromatography is also useful in purification
procedures.

[0122]Proteins and peptides may be modified using ordinary molecular
biological techniques to improve their resistance to proteolytic
degradation or to optimize solubility properties or to render them more
suitable as a therapeutic agent. Analogs of such polypeptides include
those containing residues other than naturally occurring L-amino acids,
e.g., D-amino acids or non-naturally occurring synthetic amino acids. The
polypeptides useful in the invention may further be conjugated to
non-amino acid moieties that are useful in their application. In
particular, moieties that improve the stability, biological half-life,
water solubility, and immunologic characteristics of the peptide are
useful. A non-limiting example of such a moiety is polyethylene glycol
(PEG).

II. Methods

[0123]The present invention provides methods for directing the
differentiation of a cell of the invention to a specific cell lineage by
overexpressing a biomarker nucleic acid in the cell. The present
invention also provides methods for directing the differentiation of a
cell of the invention to a specific cell lineage by overexpressing a
biomarker protein in the cell.

[0124]In one embodiment, the differentiation of a cell of the invention is
directed to the myeloid lineage. In another embodiment of the invention,
the differentiation of a cell of the invention is directed to the
lymphoid lineage.

[0125]One embodiment of the invention comprises directing the
differentiation of a cell of the invention into a natural kill (NK) cell
by overexpressing a nucleic acid encoding Zinc finger protein (Zfp) 105
the cell. Another embodiment of the invention comprises directing the
differentiation of a cell of the invention into a NK cell by
overexpressing Zfp105 protein in the cell.

[0126]Another embodiment of the invention comprises directing the
differentiation of a cell of the invention into a monocyte cell by
overexpressing a nucleic acid encoding Ets2 in the cell. Another
embodiment of the invention comprises directing the differentiation of a
cell of the invention into a monocyte cell by overexpressing Ets2 protein
in the cell.

[0127]Yet another embodiment of the invention comprises directing the
differentiation of a cell of the invention into a B-cell by
overexpressing a nucleic acid encoding Chd7 in the cell. Another
embodiment of the invention comprises directing the differentiation of a
cell of the invention into a B-cell by overexpressing Chd7 protein in the
cell.

[0128]One embodiment of the invention comprises directing the
differentiation of a cell of the invention into a B-cell by
overexpressing a nucleic acid encoding EDAR in the cell. Another
embodiment of the invention comprises directing the differentiation of a
cell of the invention into a B-cell by overexpressing EDAR protein in the
cell.

[0129]Still another embodiment of the invention comprises directing the
differentiation of a cell of the invention to a myeloid lineage by
overexpressing a nucleic acid encoding Med8 in the cell. Another
embodiment of the invention comprises directing the differentiation of a
cell of the invention to a myeloid lineage by overexpressing Med8 protein
in the cell.

[0130]One embodiment of the invention comprises directing the
differentiation of a cell of the invention to a myeloid lineage by
overexpressing a nucleic acid encoding Med14 in the cell. Another
embodiment of the invention comprises directing the differentiation of a
cell of the invention to a myeloid lineage by overexpressing Med14
protein in the cell.

[0131]Another embodiment of the invention comprises directing the
differentiation of a cell of the invention to a myeloid lineage by
overexpressing a nucleic acid encoding Glis2 in the cell. Another
embodiment of the invention comprises directing the differentiation of a
cell of the invention to a myeloid lineage by overexpressing Glis2
protein in the cell.

[0132]One embodiment of the invention comprises directing the
differentiation of a cell of the invention into a B-cell by
overexpressing a nucleic acid encoding Dzip1 in the cell. Another
embodiment of the invention comprises directing the differentiation of a
cell of the invention into a B-cell by overexpressing Dzip1 protein in
the cell.

[0133]Another embodiment of the invention comprises directing the
differentiation of a cell of the invention to a myeloid lineage by
overexpressing a nucleic acid encoding Tnfaip8l1 in the cell. Another
embodiment of the invention comprises directing the differentiation of a
cell of the invention to a myeloid lineage by overexpressing Tnfaip8l1
protein in the cell.

[0134]One embodiment of the invention comprises directing the
differentiation of a cell of the invention into a B-cell by
overexpressing a nucleic acid encoding 2210016F16Rik in the cell. Another
embodiment of the invention comprises directing the differentiation of a
cell of the invention into a B-cell by overexpressing 2210016F16Rik
protein in the cell.

[0135]One embodiment of the invention comprises directing the
differentiation of a cell of the invention into a B-cell by
overexpressing a nucleic acid encoding Tbl1x (transducin (beta)-like 1
X-linked) in the cell. Another embodiment of the invention comprises
directing the differentiation of a cell of the invention into a B-cell by
overexpressing Tbl1x protein in the cell.

[0136]Another embodiment of the invention comprises directing the
differentiation of a cell of the invention to a myeloid lineage by
overexpressing nucleic acid encoding Mina (myc induced nuclear antigen)
in the cell. Another embodiment of the invention comprises directing the
differentiation of a cell of the invention to a myeloid lineage by
overexpressing Mina protein in the cell.

[0137]In one embodiment of the invention, a biomarker nucleic acid is
administered to a mammal whereby the biomarker nucleic acid enters a cell
of the invention, is expressed therein, and thereby directs the cell's
differentiation. In another embodiment of the present invention, a
biomarker protein is administered to a mammal whereby the biomarker
protein contacts or is introduced to a cell of the invention, thereby
directing the cell's differentiation. In one embodiment the mammal is a
human.

[0138]In another embodiment of the present invention, a cell of the
invention is harvested from a mammal and brought into contact with a
biomarker nucleic acid whereby the biomarker nucleic acid directs the
cell's differentiation to a cell lineage. In another embodiment of the
present invention, a cell of the invention is harvested from a mammal and
brought into contact with a biomarker protein whereby the biomarker
protein directs the cell's differentiation to a cell lineage. A cell of
the invention may be cultured, expanded and allowed to proliferate and
differentiate ex vivo prior to being returned to the mammal. In one
embodiment, the mammal is a human.

[0139]The culture of a cell of the invention preferably occurs under
conditions that increase the number of such cells and/or the colony
forming potential of such cells. The conditions used refer to a
combination of conditions known in the art (e.g., temperature, CO2
and O2 content, nutritive media, etc.). The time sufficient to
increase the number of cells is a time that can be easily determined by a
person skilled in the art, and can vary depending upon the original
number of cells seeded. As an example, discoloration of the media can be
used as an indicator of confluency. Additionally, different volumes of
the blood product can be cultured under identical conditions, and cells
can be harvested and counted over regular time intervals, thus generating
the "control curves." These "control curves" can be used to estimate cell
numbers in subsequent occasions.

[0140]The conditions for determining colony forming potential are
similarly determined. Colony forming potential is the ability of a cell
to form progeny. Assays for this are well known to those of ordinary
skill in the art and include seeding cells into a semi-solid substrate or
media, treating them with growth factors and counting the number of
colonies.

[0141]In all of the culturing methods according to the invention, except
as otherwise provided, the liquid cell culture media utilized herein are
conventional media for culturing cells. Examples include, but are not
limited to, RPMI, DMEM, ISCOVES, etc. Typically these media are
supplemented with human or animal plasma or serum. Such plasma or serum
can contain small amounts of growth factors. The media used according to
the present invention, however, can depart, in certain embodiments, from
that used conventionally in the prior art. In particular, a cell of the
invention can be cultured on matrices and devices for extended periods of
time without the need for adding any exogenous growth agents (other than
those which may be contained in plasma or serum, hereinafter "serum"),
without inoculating the environment of the culture with stromal cells and
without using stromal cell conditioned media. In a continuous cell
culture system, it is preferred to add and remove the cell culture media
from the vessel at a rate of less than 1/2 volume change per day.

[0142]The growth agents of particular interest in connection with the
present invention are hematopoietic growth factors. By hematopoietic
growth factors, it is meant factors that influence the survival,
proliferation or differentiation of hematopoietic cells. Growth agents
that affect only survival and proliferation, but are not believed to
promote differentiation, include but are not limited to the interleukins
3, 6 and 11, stem cell ligand and FLT-3 ligand. Hematopoietic growth
factors that promote differentiation include but are not limited to the
colony stimulating factors such as GMCSF, GCSF, MCSF, Tpo, Epo,
Oncostatin M, and interleukins other than IL-3, 6 and 11. The foregoing
factors are well known to those of ordinary skill in the art. Most are
commercially available. They can be obtained by purification, by
recombinant methodologies or can be derived or synthesized synthetically.

[0143]It is possible according to the invention to preserve a cell of the
invention and to stimulate the expansion and differentiation of a cell of
the invention using the compositions and methods of the present
invention. Once a cell of the invention has been contacted with a
biomarker nucleic acid or a biomarker protein to direct differentiation
of the cell to a particular cell lineage, the cell can be expanded and,
for example, be specifically identified and/or selected by the biomarkers
described herein before being returned to the body to supplement,
replenish, or restore a patient's myeloid or lymphoid cell populations.
This might be appropriate, for example, after an individual has undergone
chemotherapy.

[0144]In another embodiment of the invention, a cell of the invention may
be cultured for an extended period of time, and aliquots of the cultured
cells are harvested spaced apart in time or intermittently. Harvesting
cells encompasses dislodging or separation of cells from the matrix. This
can be accomplished using a number of methods, such as enzymatic,
centrifugal, electrical or by size, or by flushing of the cells using the
media in which the cells are incubated. The cells can be further
collected and separated. "Harvesting steps spaced apart in time" or
"intermittent harvest of cells" is meant to indicate that a portion of
the cells are harvested, leaving behind another portion of cells for
their continuous culture in the established media, maintaining a
continuous source of the original cells and their characteristics.
Harvesting "at least a portion of" means harvesting a subpopulation of or
the entirety of. Thus, as will be understood by one of ordinary skill in
the art, the invention can be used to expand the number of cells, all the
while harvesting portions of those cells being expanded for treatment to
develop even larger populations of differentiated cells.

[0145]The present invention contemplates the use of the compositions and
methods of the invention in the treatment of a subject, preferably a
mammal, more preferably a human, suffering from a disease or disorder
wherein the directed differentiation of a cell of the invention would be
beneficial the subject. The term "beneficial" as used herein, refers to a
reduction in the severity or frequency of symptoms, a restoration of
health, or a therapeutic improvement. Some non-limiting examples where
the compositions and methods of the present invention are useful include
treating any disease or disorder where the blood cell count of a subject
is dysregulated, includin, but not limited to diseases and disorders
where blood cells are depleted, such as anemia, destroyed by a pathogen,
such as Aquired Immunodeficiency Syndrome (AIDS), or where hematopoiesis
is dysregulated, such as in cancer (e.g. leukemias and lymphomas). Also
contemplated is the use of the compositions and methods of the present
invention in situations where a subject's blood cell count is
dysregulated due to a therapeutic treatment or regimine, such as
chemotherapy or radiation therapy.

Pharmaceutical Compositions and Methods of Use

[0146]Compositions comprising biomarker nucleic acids or biomarker
proteins can be incorporated into pharmaceutical compositions suitable
for administration. As used herein the language "pharmaceutically
acceptable carrier" is intended to include any and all solvents,
dispersion media, coatings, antibacterial and antifungal agents, isotonic
and absorption delaying agents, and the like, compatible with
pharmaceutical administration. Supplementary active compounds can also be
incorporated into the compositions.

[0147]A formulated composition comprising a biomarker nucleic acid or
biomarker protein can assume a variety of states. In some examples, the
composition is at least partially crystalline, uniformly crystalline,
and/or anhydrous (e.g., less than 80, 50, 30, 20, or 10% water). In
another example, the biomarker nucleic acid or biomarker protein is in an
aqueous phase, e.g., in a solution that includes water, this form being
the preferred form for administration via inhalation.

[0148]The aqueous phase or the crystalline compositions can be
incorporated into a delivery vehicle, e.g., a liposome (particularly for
the aqueous phase), or a particle (e.g., a microparticle as can be
appropriate for a crystalline composition). Generally, the composition
comprising a biomarker nucleic acid or biomarker protein is formulated in
a manner that is compatible with the intended method of administration.

[0149]A biomarker nucleic acid or biomarker protein preparation can be
formulated in combination with another agent, e.g., another therapeutic
agent or an agent that stabilizes a biomarker nucleic acid or biomarker
protein agent, e.g., a protein that complexes with the biomarker nucleic
acid or biomarker protein agent. Still other agents include chelators,
e.g., EDTA (e.g., to remove divalent cations such as Mg2+), salts,
RNAse inhibitors (e.g., a broad specificity RNAse inhibitor such as
RNAsin) and so forth.

[0150]In one embodiment, the biomarker nucleic acid or biomarker protein
agent is administered in conjunction with another therapeutic agent such
as an antibiotic, an antiviral, an anti-inflammatory, a chemotherapeutic
agent, a pain relieving agent, or any other therapeutic compound useful
in the treatment of a particular disease or disorder. The biomarker
nucleic acid or biomarker protein agent may be administered as part of an
on-going treatment regimen or therapy for a particular disease such as
chemotherapy or radiation therapy for various cancers.

[0151]Pharmaceutical compositions of the invention include a
pharmaceutical carrier that may contain a variety of components that
provide a variety of functions, including regulation of drug
concentration, regulation of solubility, chemical stabilization,
regulation of viscosity, absorption enhancement, regulation of pH, and
the like. The pharmaceutical carrier may comprise a suitable liquid
vehicle or excipient and an optional auxiliary additive or additives. The
liquid vehicles and excipients are conventional and commercially
available. Illustrative thereof are distilled water, physiological
saline, aqueous solutions of dextrose, and the like. For water soluble
formulations, the pharmaceutical composition preferably includes a buffer
such as a phosphate buffer, or other organic acid salt, preferably at a
pH of between about 7 and 8. For formulations containing weakly soluble
antisense compounds, micro-emulsions may be employed, for example by
using a nonionic surfactant such as polysorbate 80 in an amount of
0.04-0.05% (w/v), to increase solubility. Other components may include
antioxidants, such as ascorbic acid, hydrophilic polymers, such as,
monosaccharides, disaccharides, and other carbohydrates including
cellulose or its derivatives, dextrins, chelating agents, such as EDTA,
and like components well known to those in the pharmaceutical sciences,
e.g., Remington's Pharmaceutical Science, latest edition (Mack Publishing
Company, Easton, Pa.).

[0152]Nucleic acid biomarkers and protein biomarkers contemplated in the
invention include the pharmaceutically acceptable salts thereof,
including those of alkaline earths, e.g., sodium or magnesium, ammonium
or NM4.sup.+, wherein X is C1-C4 alkyl. Other
pharmaceutically acceptable salts include organic carboxylic acids such
as acetic, lactic, tartaric, malic, isethionic, lactobionic, and succinic
acids; organic sulfonic acids such as methanesulfonic, ethanesulfonic,
and benzenesulfonic; and inorganic acids such as hydrochloric, sulfuric,
phosphoric, and sulfamic acids. Pharmaceutically acceptable salts of a
compound having a hydroxyl group include the anion of such compound in
combination with a suitable cation such as Na.sup.+, NH4.sup.+, or
the like.

[0154]Suitable methods for nucleic acid delivery according to the present
invention are believed to include virtually any method by which a nucleic
acid (e.g., DNA, RNA, including viral and nonviral vectors) can be
introduced into an organelle, a cell, a tissue or an organism, as
described herein or as would be known to one of ordinary skill in the
art. For example, the biomarker nucleic acid can be administered to the
subject either as a naked oligonucleotide agent, in conjunction with a
delivery reagent, or as a recombinant plasmid or viral vector which
expresses the oligonucleotide agent.

[0155]In addition to administration with conventional carriers, the
biomarker nucleic acid may be administered by a variety of specialized
delivery techniques.

[0156]Sustained release systems suitable for use with the pharmaceutical
compositions of the invention include semi-permeable polymer matrices in
the form of films, microcapsules, or the like, comprising polylactides,
copolymers of L-glutamic acid and gamma-ethyl-L-glutamate,
poly(2-hydroxyethyl methacrylate), and like materials, e.g., Rosenberg et
al., International application PCT/US92/05305.

[0157]The biomarker nucleic acid or biomarker protein agent may be
encapsulated in liposomes for therapeutic delivery, as described for
example in Liposome Technology, Vol. II, Incorporation of Drugs,
Proteins, and Genetic Material, CRC Press. The biomarker nucleic acid or
biomarker protein agent, depending upon its solubility, may be present
both in the aqueous layer and in the lipidic layer, or in what is
generally termed a liposomic suspension. The hydrophobic layer, generally
but not exclusively, comprises phospholipids such as lecithin and
sphingomyelin, steroids such as cholesterol, ionic surfactants such as
diacetylphosphate, stearylamine, or phosphatidic acid, and/or other
materials of a hydrophobic nature.

[0158]The biomarker nucleic acid or biomarker protein agent may be
conjugated to poly(L-lysine) to increase cell penetration. Such
conjugates are described by Lemaitre et al., Proc. Natl. Acad. Sci. USA,
84, 648-652 (1987). The procedure requires that the 3'-terminal
nucleotide be a ribonucleotide. The resulting aldehyde groups are then
randomly coupled to the epsilon-amino groups of lysine residues of
poly(L-lysine) by Schiff base formation, and then reduced with sodium
cyanoborohydride. This procedure converts the 3'-terminal ribose ring
into a morpholine structure antisense oligomers.

[0159]A biomarker nucleic acid or biomarker protein agent of the invention
also includes conjugates with appropriate ligand-binding molecules. The
biomarker nucleic acid or biomarker protein agent may be conjugated for
therapeutic administration to ligand-binding molecules which recognize
cell-surface molecules, such as according to International Patent
Application WO 91/04753. The ligand-binding molecule may comprise, for
example, an antibody against a cell surface antigen, an antibody against
a cell surface receptor, a growth factor having a corresponding cell
surface receptor, an antibody to such a growth factor, or an antibody
which recognizes a complex of a growth factor and its receptor. Methods
for conjugating ligand-binding molecules to oligonucleotides are detailed
in WO 91/04753.

[0160]An pharmaceutical composition comprising a biomarker nucleic acid or
biomarker protein of the invention can be administered at a unit dose
less than about 75 mg per kg of bodyweight, or less than about 70, 60,
50, 40, 30, 20, 10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, or
0.0005 mg per kg of bodyweight, and less than 200 nmol of a biomarker
nucleic acid or biomarker protein per kg of bodyweight, or less than
1500, 750, 300, 150, 75, 15, 7.5, 1.5, 0.75, 0.15, 0.075, 0.015, 0.0075,
0.0015, 0.00075, 0.00015 nmol of a biomarker nucleic acid or biomarker
protein per kg of bodyweight. It is understood that any and all whole or
partial integers between the ranges set forth here are included herein.
The unit dose, for example, can be administered by injection (e.g.,
intravenous or intramuscular, intrathecally, or directly into an organ),
inhalation, or a topical application.

[0161]Delivery of an a biomarker nucleic acid or biomarker protein
directly to an organ can be at a dosage on the order of about 0.00001 mg
to about 3 mg per organ, or preferably about 0.0001-0.001 mg per organ,
about 0.03-3.0 mg per organ, about 0.1-3.0 mg per organ or about 0.3-3.0
mg per organ.

[0162]In one embodiment, the unit dose is administered less frequently
than once a day, e.g., less than every 2, 4, 8 or 30 days. In another
embodiment, the unit dose is not administered with a frequency (e.g., not
a regular frequency). For example, the unit dose may be administered a
single time. Alternatively, it is possible to administer the composition
with a frequency of less than once per day, or, for some instances, only
once for the entire therapeutic regimen.

[0163]In one embodiment, a subject is administered an initial dose, and
one or more maintenance doses of a biomarker nucleic acid or biomarker
protein. The maintenance dose or doses are generally lower than the
initial dose, e.g., one-half less of the initial dose. A maintenance
regimen can include treating the subject with a dose or doses ranging
from 0.01 μg to 75 mg/kg of body weight per day, e.g., 70, 60, 50, 40,
30, 20, 10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, or 0.0005 mg per
kg of bodyweight per day. The maintenance doses are preferably
administered no more than once every 5, 10, or 30 days. Further, the
treatment regimen may last for a period of time which will vary depending
upon the nature of the particular disease, its severity and the overall
condition of the patient. In preferred embodiments the dosage may be
delivered no more than once per day, e.g., no more than once per 24, 36,
48, or more hours, e.g., no more than once every 5 or 8 days. Following
treatment, the patient can be monitored for changes in his condition and
for alleviation of the symptoms of the disease state. The dosage of the
compound may either be increased in the event the patient does not
respond significantly to current dosage levels, or the dose may be
decreased if an alleviation of the symptoms of the disease state is
observed, if the disease state has been ablated, or if undesired
side-effects are observed.

[0164]The effective dose can be administered in a single dose or in two or
more doses, as desired or considered appropriate under the specific
circumstances. If desired to facilitate repeated or frequent infusions,
implantation of a delivery device, e.g., a pump, semi-permanent stent
(e.g., intravenous, intraperitoneal, intracistemal or intracapsular), or
reservoir may be advisable.

[0165]Certain factors may influence the dosage required to effectively
treat a subject, including but not limited to the severity of the disease
or disorder, previous treatments, the general health and/or age of the
subject, and other diseases present. It will also be appreciated that the
effective dosage of a biomarker nucleic acid or biomarker protein used
for treatment may increase or decrease over the course of a particular
treatment. Changes in dosage may result and become apparent from the
results of diagnostic assays. For example, the subject can be monitored
after administering a biomarker nucleic acid or biomarker protein
composition. Based on information from the monitoring, an additional
amount of biomarker nucleic acid or biomarker protein composition can be
administered.

[0166]Dosing is dependent on severity and responsiveness of the disease
condition to be treated, with the course of treatment lasting from
several days to several months, or until a cure is effected or a
diminution of disease state is achieved. Optimal dosing schedules can be
calculated from measurements of drug accumulation in the body of the
patient. Persons of ordinary skill can easily determine optimum dosages,
dosing methodologies and repetition rates. Optimum dosages may vary
depending on the relative potency of individual compounds, and can
generally be estimated based on EC50s found to be effective in in
vitro and in vivo animal models.

[0167]A pharmaceutical composition of the invention may further comprise
one or more additional pharmaceutically active agents. The compositions
and methods of the present invention can be used in combination with
other treatment regimens, including virostatic and virotoxic agents,
antibiotic agents, antifungal agents, anti-inflammatory agents, as well
as combination therapies, and the like. The invention can also be used in
combination with other treatment modalities, such as chemotherapy,
cryotherapy, hyperthermia, radiation therapy, and the like.

Experimental Examples

[0168]The invention is further described in detail by reference to the
following experimental examples. These examples are provided for purposes
of illustration only, and are not intended to be limiting unless
otherwise specified. Thus, the invention should in no way be construed as
being limited to the following examples, but rather, should be construed
to encompass any and all variations which become evident as a result of
the teaching provided herein.

[0169]The materials and methods employed in the experiments disclosed
herein are now described.

Mice and Cell Purification

[0170]All cell types were purified according to published protocols using
a combination of magnetic and flow cytometric cell sorting to achieve at
least 95% purity. C57BV6-CD45.1 mice were housed in a specific pathogen
free barrier and fed autoclaved acidified water and mouse chow ad
libitum. All cells were purified from 8-1 2 week-old female mice; each
purification involved pools of tissue from at least 4 mice, and each
population was purified on two separate occasions and applied to
independent arrays (biological replicates). RNA from all samples was
processed together and amplified from approximately the same number of
cells prior to hybridization to Affymetrix MOE430 2.0 microarrays. These
arrays include ˜45,000 probe sets that, due to probe set
redundancy, represent about two-thirds of the known coding genome. Each
cell population was isolated from whole bone marrow (WBM), splenocytes,
or peripheral blood (PB) of four mice. WBM was isolated by flushing the
tibias and femurs with Hank's Balanced Salt Solution (HBSS; Invitrogen).
Spleens were collected, placed in HBSS, crushed, and filtered in order to
obtain splenocytes. PB was collected by retro-orbital bleeds and placed
in 2% Dextran T500 in PBS with IOU of heparin. PB was allowed to sit for
30 minutes and the top layer was collected. Cells were then resuspended
at 1×108 cells/ml and stained on ice for 15 minutes with
population-specific antibody cocktails followed by an HBSS wash. They
were then isolated via a triple-laser instrument (MoFlow, Cytomation).
All antibodies were obtained from BD Pharmigen unless otherwise stated.

[0171]Erythrocytes, granulocytes, and LT-HSCs were isolated from WBM.
Nucleated erythrocytes were Ter-119.sup.+, CD3.sup.-, CD4.sup.-,
CD8.sup.-, Mac-1.sup.-, Gr-1.sup.-, and B220.sup.-. Granulocytes were
Gr-1.sup.+, clone 7/4.sup.+ (Cedarlane Labs), CD2.sup.-, CD5.sup.-,
B220.sup.-, F4/80.sup.- (eBiosciences), ICAM-I.sup.-, Ter-119.sup.-.
LT-HSCs were isolated. Briefly, WBM was stained with Hoescht 33342 and
the Sca-1.sup.+ cells were enriched by magnetic separation, followed by
flow cytometry for sidepopulation (SP) and Sca-1.sup.+, c-it.sup.+, and
Lin.sup.- (Mac-1, Gr-1, Ter119, B220, CD4, CD8 (eBiosciences). T-cells
and B-cells were isolated from splenocytes. Naive helper T-cells were
freshly isolated CD.sup.+, CD25.sup.-, CD69.sup.- cells. Naive cytotoxic
T-cells were CD4.sup.+, CD25.sup.-, and CD69.sup.-. Splenocytes enriched
for naive T-cells were isolated and treated with concanavalin A (1 pg 1
ml, Sigma) to obtain activated T-cells. Eight to eleven hours after ConA
stimulation, activated T-cells were isolated by sorting for the markers
CD25.sup.+ and CD69 B.sup.+. B-cells were CD19.sup.+ and 33D1.sup.splenocytes. Monocytes were isolated from PB based on Forward and Side
Scatter properties as well as being Mac-1.sup.+. NK cells were isolated
from spleen, as Nk1.1.sup.+ and CD3.sup.- cells.

RNA Purification and Hybridization to Microarrays

[0172]RNA was isolated from 1×105 cells (except for HSCs which
was from 2.5-5×104 cells), using the RNAqeuous kit (Ambion,
Austin, Tex., USA), treated with DNAseI, and precipitated with
phenol:chloroform:isoamyl alcohol. The RNA was linearly amplified as
previously reported. Briefly, two rounds of T7-based in vitro
transcription using the MessageAmp kit (Ambion) was undertaken and the
RNA was labeled with biotin-conjugated UTP and CTP (Enzo Biotech).
Amplified biotinylated RNA (20 ug) was diluted in fragmentation buffer,
incubated at 94° C. for 25 minutes, and stored at -80° C. A
sample was run on a 4% agarose gel to confirm an RNA fragment length of
approximately 50 bp. The labeled RNA was hybridized to MOE430.2 chips
according to standard protocols. For signal amplification, the chips were
washed and counterstained with PE-conjugated strepavidin and a
biotinylated anti-streptavidin antibody. The raw image and intensity
files were generated using GCOS 1.0 software (Affymetrix). All
microarrays used in this study had to pass several quality control tests
including a scale factor<5, a 5' to 3' probe ratio<20, and
replicate correlation coefficient>0.96.

Microarray Analysis

[0173]Normalization and model-based expression measurements were performed
with GC-RMA. GC-RMA, as well as additional analytical and annotation
packages, are available as part of the open-source Bioconductor project
(www.bioconductor.org) within the statistical programming language R
(www.cran.r-project.org). Cluster and Principle Components Analyses were
performed with the R base package. Lineage fingerprints were established
by setting on/off expression thresholds (Off<4, On>5) and using
Boolean logic to find uniquely expressed genes in each cell type and each
grouping. When selecting a T-cell fingerprint, activated T-cells were
removed from the data set and naive T-cells (both CD4.sup.+ and
CD8.sup.+) were averaged together.

Knockout Analysis

[0174]All knockout data were obtained via the 3.44 MGI database
(www.informatics.jax.org). Knockouts with a reported hematopoietic or
immune phenotype were grouped into a single list (`hematopoietic knockout
data`). All genes on the array that have a reported knockout were
identified. The frequency of fingerprint genes that have a hematopoietic
phenotype was compared to the frequency of hematopoietic knockouts found
by chance within all knock-out data. A Z-statistic was used to determine
the significance of enrichment fingerprint genes found within the
knockouts with a reported hematopoietic phenotype. A Z-statistic above 3
S.D. was considered significant for this enrichment.

KEGG Analysis

[0175]KEGG is a suite of gene databases systematically organized into
metabolic and signaling pathways (www.genome.ad.jp/kegg/). The mean
expression value for each hematopoietic cell type was obtained for all
KEGG pathways that contained genes found on the microarray. Pathways with
a significant variation between the cell types (ANOVA
p-value≦0.05) were identified (65 significant pathways). The
pathways were then ranked by maximal abundance for each cell type and a
heat map was generated using the centered data.

Chromosomal Analysis

[0176]Affymetrix probes were assigned a value of 1 if they were equal to
or greater than 4.5 and 0 if they were below 4.5. Each probe was then
plotted to its chromosomal position and a chromosomal expression map was
created for each chromosome for each cell type by an R function we
developed. Briefly, this function uses a sliding and overlapping window,
which covers 20 probe sets (representing 14 genes on average) at one time
and overlaps with the previous nineteen, that takes the sum of 1's within
the window for the y-axis and the mean chromosomal position for the
x-axis and uses these values to plot the expression map. For comparison
between cell types, the expression map of one cell type was directly
subtracted from all the other cell types in turn. To quantitate openness
of chromatin, the area under the curves was calculated and divided by the
possible area under the curve (if all y-values were 20) to calculate a
percentage of more open chromatin as shown by the chromosomal expression
maps.

[0178]The results of the experiments presented in this Example are now
described.

Example 1

Gene Expression Patterns Reflect Ontogeny

[0179]To identify genes uniquely expressed in HSC and their differentiated
progeny, a global gene expression profiling approach was used to
determine in parallel the transcribed genome of known coding transcripts
in freshly isolated HSC as well as the major differentiated hematopoietic
lineages, including natural killer (NK) cells, T-cells, B-cells,
monocytes, neutrophils, and nucleated erythrocytes. Both activated and
naive CD4+ (helper) and CD8+ (cytotoxic) T-cell subsets were examined.
Each population was purified to at least 95% purity, and multiple
parameters were standardized to reduce technical variation. RNA from the
samples was processed and hybridized to Affymetrix MOE430 2.0
microarrays, which have probe sets representing about 20,000 genes
(˜2/3 of the known coding genome). The correlation coefficients
between chip pairs were at least 0.96, indicating very high data quality.
This is the first study to interrogate a stem cell and multiple specific
progeny types.

[0180]The data set was first used to investigate assumptions concerning
relatedness of the hematopoietic cell types, as the similarity of gene
expression profiles between different populations should reflect their
ontogeny relationships, and perhaps shed light on the debate regarding
the developmental origins of the erythroid branch. Cluster analysis was
used to assess relative distance of the transcriptome of each cell type,
resulting in a branched "family tree" based on the similarity between
overall transcription patterns (FIG. 1A). HSC clustered with the
lymphocytes, suggesting a strong similarity in their transcriptional
activity. This observation supports a recent report that HSC and
lymphocytes share many similarities, including cell surface activation
molecules, and may indicate a conserved mechanism between HSC and
lymphocytes of long term quiescence interrupted by bursts of
proliferative stimuli. Also noteworthy is the very distinct nature of
activated T-cells (both CD4+ and CD8+).

[0181]Principle components analysis (PCA) was used to further explore the
cell-type relationships. When the relative expression distance between
each averaged chip pair was examined with PCA in two dimensions (first
and second principle components; PC1, PC2), the cell types cluster on the
basis of ontogeny in the hematopoietic tree, with the stem cell having a
centralized position (FIG. 1B). This analysis reveals the striking
segregation of lymphoid, myeloid, and erythroid cells into three discrete
clusters. Both cluster analysis and PCA indicate that erythrocytes are
distant from myeloid cells (monocytes, granulocytes), supporting a recent
observation that they arise independently of a common myeloid progenitor.

Experiment #2: Genetic Fingerprints Unique to Each Cell Type

[0182]The data set was next examined to determine whether there were genes
exclusively expressed by particular cell types. Such a genetic
"fingerprint" would be expected to contain some genes encoding proteins
involved in functions unique to that cell type, as well as regulatory
proteins that may direct expression of other genes in the fingerprint.

[0183]"Fingerprint genes," as used herein, refer to those that were
observed to be expressed in one cell type, but not expressed in all other
profiles. Expression thresholds above and below a conservative window
were selected by real-time PCR. An expression value of 5 or greater was
considered to be expressed in that cell type, and 4 or less was
considered to be not expressed in that cell type (log(2) scale). Using
these thresholds to filter the data, unique genes expressed in each cell
type were identified. Examples of genes found in fingerprints are
ecotropic viral integration site-1 (Evi1; a known proto-oncogene), one of
the most differentially expressed genes in HSC, killer cell lectin-like
receptor family E member-1 (Klre1; a known NK cell receptor), and T cell
receptor alpha on T cells (FIG. 2A).

[0184]The filter was also applied to groups of cell-types to determine a
"shared" fingerprint for all differentiated cells, as well as the
lymphoid (NK cells, T-cells, B-cells) and myeloid (monocytes and
granulocytes) branches, which defined mutually expressed genes excluded
from other cells (FIG. 2B). Strikingly, expression of the gene for the
cell surface marker CD48 identifies all the differentiated cell types but
not HSC, consistent with recent reports and antibody staining (not
shown). Similarly, CD2 expression marks the lymphoid lineage, and Trem1
the myeloid lineage. Due to the similarities of naive CD4+ and CD8+
T-cells and their activated counterparts, for the majority of these
analyses naive T-cells were used to represent the T-cell lineage. The
relationships between the T-cell sub-types are dissected further below.

[0185]Each cell type fingerprint contained 40-100 genes, except the HSC
and erythroid cells, which both had ˜350 genes (FIG. 2C). The
smallest fingerprint was that of the T-cells, which had only 42 unique
genes, due to the similarities between T-cells and other lymphoid cells.
The NK and B-cell fingerprints were each around 100 genes. The shared
lymphoid fingerprint, representing genes shared by all lymphoid cells and
excluded from stem cells, contained only 18 genes, reflecting their
similarity to stem cells. The myeloid fingerprint contained 154 genes,
and the differentiated fingerprint, which includes all differentiated
cell types and excludes stem cells, contains 36 genes (FIG. 2D). Finally,
genes expressed in all cell types examined were also identified to
establish a common hematopoietic signature of ˜9000 genes. Many of
these are likely house-keeping genes shared with many non-hematopoietic
cell types, but some are unique to the entire hematopoietic system, such
as CD45.

[0186]The genetic fingerprints contain some genes known to be important
for the function of certain cell types, such as Gatal in the erythroid
lineage and the CSF1 receptor (Csf1r) in the monocyte lineage, validating
the approach for identification of cell-type-specific genes (FIG. 3A).
The fingerprints also contain many under-studied genes that may have
regulatory role or may serve as novel markers for those cell types.
Likewise, the shared fingerprint genes are either required for the
function of all family cell types, such as Atm in the lymphoid
fingerprint, or for differentiation of the cell-type family (FIG. 3A).
The progenitors of differentiated branches are likely to express some of
the shared fingerprint genes (FIG. 3A and FIG. 3B), which could be
developed as tools for markers of those progenitors, or as regulators of
their fate.

[0187]To assess whether deficiencies of fingerprint genes result in
hematopoietic cell defects, reported phenotypes in the Jax Mouse Genome
Informatics database (v.3.44) were examined for all genes that had a
reported knock-out allele. Remarkably, several of the fingerprint genes
with available knock-outs show serious defects in either differentiation
or functionality of a lineage (FIG. 3C, Table 1). A striking example from
the B-cell fingerprint is the Ebf1 knockout mouse, which exhibits a
complete loss of B-cells. Likewise, ablation of HoxA9, an HSC fingerprint
gene, results in severely impaired HSC. All fingerprints, with one
exception, demonstrated a statistically significant enrichment of genes
exhibiting a defect in development or function of hematopoietic cells
when knocked-out (z-test). Only the whole common fingerprint, which
contains all genes expressed in common in all ten populations, was not
found to be enriched for a hematopoietic knock-out phenotype, most likely
due to the preponderance of house-keeping genes within this list.

Experiment #3: HSC and T-Cells Share a Similar Transcriptional Program of
Activation.

[0188]Observations have suggested that HSC and T-cells share a number of
markers as well as regulatory genes. These similarities could result from
a similar life cycle of long-term quiescence followed by expansion, or
this could be related to the evolutionary history of the hematopoietic
system.

[0189]Cluster analysis was repeated with T-cell data alone. This analysis
again showed that the activation state is far more important to
distinguish T-cell sub-types than their CD4/CD8 status as helper or
effector T-cells (FIG. 4A). A comparison of the activated and naive
T-cell gene expression data to previous study in which genes were
identified that change with HSC activation, as stimulated by
5-fluorouracil treatment, identified genes that differ between activated
and naive T-cells, generated from a pair-wise comparison. The expression
pattern of these genes in HSC over the activation time-course was
examined, and the average expression values plotted. On average, genes
that were upregulated in activated T-cells also showed a pattern of
significant upregulation in activated HSC (FIG. 4B; genes increasing in
expression on days 2-6 of HSC activation; one-way ANOVA
p≦5.23×104, alpha=0.05), whereas naive T-cell genes
have the reciprocal signature, being more highly expressed in quiescent
HSC (p≦3.44×10-3, alpha=0.05). Thus, quiescent HSC are
more similar to naive T-cells, and proliferating HSC more similar to
activated T-cells suggesting that the mechanisms that regulate the
activation state in HSC and T-cells are similar.

[0190]Genes distinguishing the T-cell subsets were next identified,
irrespective of their expression pattern in the other hematopoietic
cells, using the same threshold criteria applied to the hematopoietic
fingerprints. For each T-cell type, the number of genes uniquely
expressed in each ranged from 13 (CD4-activated) to 73 (CD4-naive) (FIG.
4C). This strategy also revealed that the number of genes shared between
activated and naive CD4+ T-cells, but excluded from their CD8+
counterparts, was only four; included is Zbtb7b, a gene that permits
selection of CD4+ T-cells in the thymus. Activated and naive CD8+ cells
shared merely eleven genes, including both the α and β chains
of CD8 (FIG. 4D). However, the CD4+ and CD8+ naive T-cells shared 215
genes with each other, while the activated cells shared 174, consistent
with the clustering behavior of these sub-types, and demonstrating the
dramatic changes both helper and effector T-cells undergo during
activation (FIG. 4A). As expected, activated T-cells upregulated
established activation markers such as I12ra (CD2 5) and Icos. This list
includes a substantial number of under-characterized genes, such as two
Riken clones confirmed by RT-PCR (3110082i17Rik and 4933403f05Rik) named
Heist (for Higher Expression in Stimulated T-cells) -1 and -2.

[0191]The Kyoto Encyclopedia of Genes and Genomes (KEGG) was used to group
genes into specific pathways in order to investigate whether certain
signaling or metabolic pathways are likely to be operating in specific
cell types. The mean expression value for all arrayed genes in each
pathway was determined for every cell type, and categories with
significant variation (i.e. difference between the cell types; 65
categories; ANOVA p-value≦0.05, alpha=0.05) were displayed (FIG.
5). Notably, genes involved in Wnt signaling were over-represented in
HSC, consistent with a putative role for this pathway in cell fate
decisions. In addition, prostaglandin and leukotriene metabolism was
found enriched in myeloid cells, and Notch signaling enriched in HSC,
activated CD8+ T-cells, and monocytes.

[0192]Myeloid cells displayed the greatest over-abundance of both active
metabolic and signaling pathways, with similar numbers of each pathway
type being up regulated. T-cells showed a pattern of increasing metabolic
pathways and decreasing signaling pathways upon activation. B-cells
showed a 3-fold greater enrichment of signaling pathways over metabolic
pathways, and NK cells exhibited a similar skewing, perhaps indicative of
their role as primary sensors of the immune system. Nucleated
erythrocytes displayed lower abundance of a number of signaling pathways
while at the same time exhibiting a greater proportion of metabolic
pathways, as would be expected during the final stages erythrocyte
differentiation represented in the purified cells. HSC showed the third
highest abundance of active pathways with signaling pathways represented
more highly than metabolic pathways, consistent with a state of receptive
readiness in the quiescent HSC, poised to differentiate into mature blood
cell types.

[0193]Stem cells have been speculated to maintain an open chromatin state,
in order to facilitate rapid access to multiple lineage differentiation
programs, with chromatin becoming more closed as differentiation occur.
This notion reflects an idea of stem cell `priming" where multipotent
progenitors may express low levels of genes necessary for multiple
lineage programs, and shut down expression as lineage choices become
restricted during differentiation, and suggests a major role for
chromatin status in maintenance of "stemness" and chromatin remodeling in
differentiation.

[0194]The small numbers of HSC that typically can be obtained prevent
direct examination of the chromatin state using traditional methods.
Thus, chromatin status was probed using high-level analysis of gene
expression data by reasoning that "open" chromatin would translate into
measurable expression by cohorts of physically adjacent genes along
chromosomes. An algorithm was created to generate a map, for each cell
type, of all transcribed genes (using 4.5 as the expression threshold),
and to identify genes within windows of at least 20 adjacent co-expressed
probe sets (representing 14 genes on average). Each cell type's
chromosomal expression map was subtracted from the chromosomal expression
maps of each other population to observe differences in chromosomal
transcribed regions, as illustrated in FIG. 6A. When HSC were compared to
all other cell types across the X-chromosome, the HSC exhibited many more
regions of open chromatin, evidenced by peaks on the plots. The reverse
is true for the erythrocytes (FIG. 6B), with mostly valleys. By
calculating the area under the curve, the percentage of open chromatin
for each cell type was determined and displayed graphically for each
chromosome (FIG. 6C). On all chromosomes except 17, the chromatin is
highly accessible in HSC, relative to the other cell types. Remarkably,
the X-chromosome displays an extreme degree of openness. The same
analysis with monocytes shows a variable degree of chromatin
accessibility, while erythrocytes are largely closed except for
chromosome 14. The marked openness of chromosome 14 in erythrocytes may
indicate the presence of a number of linked genes involved in erythrocyte
generation, although it is not clear what these might be.

[0195]As shown above, each cell-type-specific fingerprint contains a
number of genes, some of which are likely to be involved in lineage
specification. To test this, the ability of overexpression of putative
regulatory fingerprint transcription factors to bias hematopoietic
differentiation was examined. Two transcription factors were tested:
Ets2, from the monocyte fingerprint representing the myeloid branch, and
zinc finger protein 105 (Zfp105), from the NK cell fingerprint
representing the lymphoid branch.

[0196]MSCV-based retroviral vectors containing each of the genes coupled
to eGFP via an IRES were used to transduce bone marrow progenitors, which
were then transplanted into lethally irradiated recipient mice (FIG. 7A).
Bone marrow transduced with a vector containing eGFP alone served as a
control. Peripheral blood of the recipients was examined 12 weeks after
transplantation for the proportion of transduced GFP+ cells in different
blood lineages, and lineage contribution in control vs. tester mice was
compared. Ets2 overexpression resulted in an overall increase in myeloid
cells, and a substantial decrease in T-cells relative to controls (FIG.
7b). When the F4/80 marker was used to specifically track monocyte
differentiation, a ˜4-fold increase in monocytes derived from
Ets2-transduced stem cells was evident in the peripheral blood, relative
to controls (two sample T-test assuming equal variances, two sided
p≦0.00 7, alpha=0.05), indicating that overexpression of Ets2 bias
differentiation toward the macrophage lineage. Overall, both T-cells and
B-cells were significantly reduced in the Zfp105 overexpressing blood
cells relative to controls (FIG. 7C). When the proportion of NK cells in
these mice was examined using the NK-specific marker NK1.1, a greater
than 5-fold increase in NK cells was observed (two sample T-test assuming
equal variances, two-sided p≦10-6, alpha=0.05).
Interestingly, the total percentage of Zfp105-transduced cells was quite
low in these mice (˜2% at 12 weeks, compared to ˜18% in
GFP-alone). This indicates that over expression of a powerful
differentiation factor is incompatible with maintenance of stem cell
functions, as required for multilineage contribution in this in vivo
assay.

[0199]Retroviral transduction of Med8 (mediator of RNA polymerase II
transcription, subunit 8 homolog), a Differentiated fingerprint gene,
skews differentiation toward the myeloid lineages. Myeloid skewing is
consistent with a role in stem cell expansion. Med8 is a putative
transcription factor that may be part of the mediator complex.

[0200]Retroviral transduction of Med14 (mediator complex subunit 14), an
HSC fingerprint gene, may induce quiescence, as seen by the low
percentage of GFP+ cells at all time points. It also skews
differentiation toward the myeloid lineages. Med14 is a putative
transcription factor that may be part of the mediator complex.

[0204]Retroviral transduction of 2210016F16Rik, an activated T-cell
fingerprint gene, skews differentiation toward the B-cell lineage.
2210016F16Rik has no known function and belongs to a group of genes
(Riken genes) identified by large scale sequencing of messenger RNA.

[0206]Retroviral transduction of Mina (myc induced nuclear antigen), an
activated T-cell fingerprint gene, skews differentiation toward the
myeloid lineages. Mina is localized to the nucleus and may play a role in
cellular proliferation.

[0207]The disclosures of each and every patent, patent application, and
publication cited herein are hereby incorporated herein by reference in
their entirety. While this invention has been disclosed with reference to
specific embodiments, it is apparent that other embodiments and
variations of this invention may be devised by others skilled in the art
without departing from the true spirit and scope of the invention. The
appended claims are intended to be construed to include all such
embodiments and equivalent variations.